A one hundred meter long ion-exchange chromatograph was used to establish rigorously the mass effects in the iron isotope fractionation in the Fe(II)–Fe(III) electron exchange equilibration. We used a highly porous, strongly basic anion exchange resin packed in glass columns. The abundance ratios of all natural iron isotopes, 54Fe, 56Fe, 57Fe, and 58Fe, in the effluent at the iron adsorption band boundary were measured with a mass spectrometer. The enrichment correlations among these isotopes were analyzed by three-isotope plots. The results clearly showed that the isotope fractionation of Fe(II)–Fe(III) is governed by the normal mass effect; the iron isotope fractionation is not proportional to the nuclear size, but proportional to the reduced mass difference of the pair of iron isotopes.